37LRP Promotes Growth and Proliferation of Gastric Cancer in Vitro and in Vivo

ORIGINAL ARTICLE MGr1-Ag/37LRP promotes growth and proliferation of gastric cancer in vitro and in vivo

L Liu1,4, L Sun2,4,KWu2, Y Shi2, Y Wang1, Y Wang3, N Zhang1, H Zhang2 and H Zhang1

Gastric carcinoma (GC) is an aggressive cancer with a poor prognosis. We previously reported that MGr1-Ag was involved in multidrug resistance and anti-apoptosis in GC. However, the exact function of MGr1-Ag in GC proliferation is not clear. In this study, we found that MGr1-Ag was highly expressed in GC tissues and four GC cell lines compared with nontumor gastric tissues or gastric epithelial mucosa cells. The high expression of MGr1-Ag/37LRP was also consistent with the decreased median survival time of GC patients. We employed lenti-mediated RNA interference technique to knock down MGr1-Ag expression in SGC7901 and MKN45 cells, respectively, and observed its effects on GC cells growth in vitro and in vivo. Further study showed that knockdown of MGr1-Ag could inhibit GC cell proliferation by inhibiting the cell cycle S-phase entry and induced apoptosis. Soft agar colony formation assay indicated that the colony formation ability of SGC7901 and MKN45 cells decreased after lenti-MGr1-Ag small interfering RNA (siRNA) infection. Western blot revealed that cyclin D1 and Bcl-2 expression were downregulated whereas p27 and Bax were upregulated in lenti-MGr-siRNA-infected GC cells. Further study demonstrated that the proliferation effect of MGr1-Ag in GC is dependent on its laminin-binding region. Taken together, these data revealed a novel function of MGr1-Ag that can possibly be used as an independent prognostic factor and a potential therapeutic target for GC.

Cancer Gene Therapy (2014) 21, 355–363; doi:10.1038/cgt.2014.36; published online 25 July 2014

INTRODUCTION critical to identify the underlying mechanism regulating prolife- Although the incidence of gastric cancer (GC) has declined in the ration in gastric cancer. West, it is a rampant health problem in many countries around the In this study, we first observed MGr1-Ag is highly expressed world. In China, more new cases of GC are diagnosed each year in GC cell lines and tissues. Subsequently, we employed the than in other countries. It is well known that GC is often diagnosed lentivirus-delivered small interfering RNA (siRNA) technique to at an advanced stage. Hence, it is helpful to fully understand its observe the effect of MGr1-Ag knockdown in human GC cell biology and behavior. Besides E-cadherin/CDH1 and TP53, a growth and proliferation in vitro and in vivo. Furthermore, we previous study by our laboratory has indicated the role of several found that resistance to cell apoptosis and induction of cell cycle tumor-suppressor genes and oncogenes in tumor development arrest, at least in part, is responsible for tumor activity of MGr1-Ag/ and progression, including RUNX3,1 p75 neurotrophin receptor 37LRP in gastric cancer. Our data indicated that MGr1-Ag/37LRP (p75 NTR) gene,2 prion,3 CIAPIN4 and MGr1-Ag/p40.5 was a potential tumor oncogene of GC and may be used as a MGr1-Ag was previously reported by our lab as an upregulated therapeutic target for gastric cancer. protein in SGC7901/VCR, a drug-resistant cell line of gastric cancer.6 Which was also termed as 37LRP and p40,7 a multi- MATERIALS AND METHODS functional protein that is required for cell differentiation, movement and growth. On the one hand, it could be exported Ethics statement to the cell surface and function as a cell surface-associated 67 kDa For tissue specimens, signed informed consent was obtained from patients laminin (LM)-binding protein. On the other hand, it has also been contributing dissected tissues. All GC cases were clinically and patho- identified as p40 ribosome-associated protein involved in tumor logically confirmed. The experimental procedures were approved by the Institutional Review Board of the Fourth Military Medical University and growth and proliferation.8 One such possibility relates to the role 9 conformed to the Helsinki Declaration and to local legislation. Animal of MGr1-Ag as a component of the protein synthetic machinery. experiments were performed in accordance with the National Guidelines kip1 Several growth regulatory genes, including cdk4, p27 and cln3, for the Care and Use of Laboratory Animals, with the approval of the are subject to translational control that satisfy the need of the Institutional Committee for Animal Research. The procedures were rapidly divided cells.10–12 Increased interest also comes from the approved by the Committee on the Ethics of Animal Experiments of the observation that the 37LRP/p40 (residues 161–180 and 205–229) Fourth Military Medical University (Xi’an, Shanxi Province, China). was shown to exhibit high LM-binding activity that is associated with poor prognosis.13,14 By a series of studies, we have demon- Cell lines and treatment conditions strated that MGr1-Ag overexpression is involved in multidrug The following human gastric adenocarcinoma cell lines were employed: resistance15 and anti-apoptosis16 in gastric cancer. Thus, it is MKN45, AGS and MKN28 were obtained from the Shanghai Cell Bank

1Department of Oncology, Tangdu Hospital, Fourth Military Medical University, Xi’an, China; 2State Key Laboratory of Cancer Biology, Institute of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an, China and 3Department of Pathology and Pathophysiology, Fourth Military Medical University, Xi’an, China. Correspondence: Professor Helong Zhang, Department of Oncology, Tangdu Hospital, Fourth Military Medical University, 1 Xinsi Road, Xi’an 710038, China. or Professor Hongbo Zhang, State Key Laboratory of Cancer Biology, Institute of Digestive Diseases Xijing Hospital, Fourth Military Medical University, No. 169, Chang le West Road, Xi’an 710032, China. E-mail: [email protected] or [email protected] 4These two authors contributed equally to this work. Received 18 February 2014; revised 25 June 2014; accepted 26 June 2014; published online 25 July 2014 MGr1-Ag can promote gastric cancer proliferation L Liu et al 356 (Shanghai, China) and SGC7901 from the Academy of Military Medical Monolayer growth assay Science (Beijing, China). The immortalized gastric epithelial mucosa cell The monolayer culture growth rate was determined using Cellomics line GES was established by the Beijing Cancer Institute (Beijing, China). Arrayscan (GeneChem). Briefly, after the cells were infected with virus for 3 Cell lines were maintained in RPMI-1640 medium containing 10% fetal days, cells of the same density were seeded into flat-bottom 96-well plates bovine serum with sodium pyruvate, nonessential amino acids, L- and grown under normal conditions. Then, the cells were observed at 0, 1, glutamine, a twofold vitamin solution (Life Technologies, Grand Island, 2, 3, 4 and 5 days using a Cellomics Arrayscan to observe the cell growth NY, USA) and a penicillin–streptomycin mixture at 37 1C in a humidified with green fluorescent protein signal. Subsequently, the growth curve of atmosphere of 5% CO2 and 95% O2. the cells was measured after each experiment. Each experiment was performed in triplicate. Tissue collection and immunochemistry A total of 80 primary GC and their noncancerous counterpart’s specimens Colony formation assay were obtained from patients who underwent surgery at the Department of A soft-agar colony formation assay was performed to assess the General Surgery in Xijing hospital from 1996 to 2004. All patients had a anchorage-independent growth ability of cells as a characteristic of well-documented clinical history, follow-up information and no preopera- in vitro tumorigenicity using a Cellomics Arrayscan (GeneChem). Briefly, tive chemotherapy and/or radiation therapy. All had undergone gastrect- SGC7901 and MKN45 cells were infected with the virus for 24 h, and the omy with lymph node dissection and were observed at Xijing Hospital cells were then detached and plated on 0.3% agarose with a 0.5% agarose through end of 2006. underlay in six-well plates (1.0 Â 104 cells per well). The number of foci Signal was detected using 3,3-diaminobenzidine as a chromogen. (4100 mm) was counted after 17 days. Each experiment was performed in Results were evaluated by a previously described formula17 in which triplicate. staining score was as follows: intensity of immunoreactivity (IR) Â the proportion of positively stained cells. IR intensity was stratified into four Cell cycle and apoptotic analysis categories: 0 to 1, weak IR; 2, moderate IR; and 3, strong IR. The percent of Flow cytometry analysis was used to determine the distribution of the cells tumor cells positive for IR was classified into four groups: 0, 0–5%; 0.33, in the cell cycle and apoptosis as previously described.19 Briefly, SGC7901 5–33%; 0.67, 33–67%; and 1, 67–100%. or MKN45 transfectant cells were seeded and infected with virus for 96 h in complete medium and then placed in a serum-free medium for 48 h. After MGr1-Ag plasmid construction and siRNA lentivirus generation collection by trypsinization, the cells were suspended in 0.5 ml of 70% 1 MGr1-Ag-targeting oligonucleotides for generating complementary DNA alcohol and kept at 4 C for 30 min. The suspension was filtered through a (cDNA) were designed from full-length MGr1-Ag by GeneChem (Shanghai, 50-mm nylon mesh, and the DNA content of the stained nuclei was China). The deletion mutants MGr1-AgDN and MGr1-AgDB were synthe- analyzed using a flow cytometer (EPICS XL; Beckman Coulter, Brea, CA, sized respectively and then subcloned into pcDNA 3.1B vector (Invitrogen, USA). Cell cycle analysis was performed using MultiCycle DNA cell cycle Carlsbad, CA, USA) with BamHI and EcoRI for producing a fusion protein analysis software (BD Biosciences, Franklin Lakes, NJ, USA). Each with C-terminal 6His tag. The siRNAs targeting MGr1-Ag were: forward: 50- experiment was performed in triplicate. Apoptotic cells were identified as a hypodiploid DNA peak that represented the cells in sub-G1. Findings cgggaGATCCTGAAGAGATTGAAATTCAAGAGATTTCAATCTCTTCAGGATCtcT 4 TTTTg-30 and reverse: 50-aattcaaaaagaGATCCTGAAGAGATTGAAATCTCTT from at least 2.0 Â 10 cells were collected and analyzed by CellQuest GAATTTCAATCTCTTCAGGATCtc-30. After testing the overexpression and software (BD Biosciences). knockdown efficiencies, stem-loop oligonucleotides were synthesized, and cloned into the lentivirus-based vector PsicoR (Addgene, Cambridge, MA, Xenograft tumor model in nude mice USA). A nontargeting stem-loop DNA PsicoR vector was generated as a Approximately 2.0 Â 106 cells were inoculated subcutaneously with 0.1 ml negative control. Lentiviral particles were prepared as described pre- of phosphate-buffered saline in the flank region of 4–6-week-old male 18 viously. GC cells were infected with siRNA lentivirus or negative control athymic nude mice (Experimental Animal Center, Fourth Military Medical virus at 7 days and examined at 10 days. GC cell lines SGC7901 and MKN45 University, Xi’an, China). At 2 weeks after tumor cell inoculation, mice were transfected with MGr1-Ag siRNA or empty vector were designated divided randomly into three groups (five mice per group) and were treated SGC7901/MGr-si, MKN45/MGr-si, SGC7901/Scr-si and MKN45/scr-si. every other day for 10 days by way of multiple-center tumor injection of 100 ml of lenti-MGr-si or Scr-si. The volume measurements of tumor were Western blotting performed once every 4 days and calculated by the formula: tumor À 2 volume ¼ length Â width Â depth Â 0.5236. Data points were expressed After pretreatment as indicated (LM pretreatment with 1 mgcm ), whole as mean tumor volume levels±s.e. The xenografted tumors were excised cells were harvested and lysed on ice for 30 min in lysis buffer (50 mM and prepared with a routine pathological procedure. Then, the tumor Tris-HCl, pH 7.4, 1% v/v Triton X-100, 1 mM EDTA, 1 mM leupeptin, sections were subjected to immunohistochemistry analysis. All animal 1mM phenylmethylsulfonyl fluoride and 10 mM NaF). Equal amounts of procedures were performed according to the guidelines of the Chinese protein (25 mgor40mg) were loaded onto a sodium dodecyl sulfate- Council on Animal Care and with appropriate institutional certification. polyacrylamide gel (8% polyacrylamide), subjected to electrophoresis at 200 V for 30 min, transferred to nitrocellulose and blocked overnight in blocking buffer (250 mM NaCl, 0.02% Tween-20, 5% goat serum and Statistical analysis 3% bovine serum albumin). For western blotting, anti-MGr1-Ag (1:100; Each experiment was repeated at least three times. Bands from western prepared by Bioss, Beijing, China), Bcl-2, Bax (1:500; Santa Cruz blot or RT-PCR analysis were quantized using Quantity One software Biotechnology, Santa Cruz, CA, USA), Caspase-3 (1:500; Abcam, Cambridge, (Bio-Rad, Hercules, CA, USA). Relative protein or mRNA levels were MA, USA) cyclin D, p27 (1:1000; Cell Signaling, Danvers, MA, USA) and calculated relative to the amount of glyceraldehyde 3-phosphate b-actin (1:5000; Sigma, St Louis, MO, USA) were added for 3 h. Filers were dehydrogenase. The difference between means was performed with washed and species-matched peroxidase-conjugated secondary antibody analysis of variance and subsequent post hoc tests. All statistical analyses was added (1:2000). Labeled bands from washed blots were detected by were performed using SPSS v11.0 software (SPSS, Chicago, IL, USA). ECL (Amersham, Piscataway, NJ, USA). Po0.05 was considered statistically significant.

Real-time reverse transcription-PCR (RT-PCR) RESULTS Total RNA was extracted with RNAzol (Biogenesis, Boulder, CO, USA) MGr1-Ag is overexpressed in GC tissues and cells according to the manufacturer’s instructions. The cDNA synthesis was The expression of MGr1-Ag/37LRP was evaluated by immuno- performed using a Moloney murine leukemia virus cDNA synthesis kit histochemistry in 80 primary GC and their noncancerous counter- (Gibco BRL, Gaithersburg, MD, USA). Taqman PCR primers were designed based on mRNA sequences using Primer Express software (Perkin-Elmer, part’s specimens (Table 1). It was found that MGr1-Ag was Santa Clara, CA, USA) supplied by Sigma Genosys Sigma (St Louis, MO, predominantly located in the cytoplasm and membrane of GC USA). Sequences were: for actin, 50-GGCGGCACCACCATGTACCCT-30 and 50- cells. In all, 12 tumors (15%) and 42 tumors (52.5%) showed weak AGGGGCCGGAC TCGTCATACT-30; for MGr1-Ag: 50-GCAGCAGGAACCCACT ( þ ) or moderate ( þþ) MGr1-Ag expression, respectively, TAGG-30 and 50-GGCAGCAGCAAACTTCAGC-30. whereas MGr1-Ag strong expression was detected in 26

Cancer Gene Therapy (2014), 355 – 363 & 2014 Nature America, Inc. MGr1-Ag can promote gastric cancer proliferation L Liu et al 357 Table 1. Patient characteristics and MGr1-Ag expression

Characteristic Total (n ¼ 80) MGr1-Ag staining P-value

Weak (IR ¼ 0–1) (n ¼ 12) Moderate (IR ¼ 2) (n ¼ 42) Strong (IR ¼ 3) (n ¼ 26)

Age o60 50 8 (16.0%) 26 (52.0%) 16 (32.0%) X60 30 4 (13.3%) 16 (53.4%) 10 (33.3%) 0.48

Gender Male 58 7 (12.0%) 30 (51.7%) 21 (36.3%) Female 22 5 (22.7%) 12 (54.6%) 5 (22.7%) 0.91

Stage (TNM)a I 31 8 (25.8%) 20 (64.5%) 3 (9.7%) II 27 4 (14.8%) 14 (51.9%) 9 (33.3%) III 15 0 (0.0%) 6 (40.0%) 9 (60.0%) IV 7 0 (0.0%) 2 (28.6%) 5 (71.4%) 0.001

Residual disease R0 44 8 (18.2%) 26 (59.1%) 10 (22.7%) R1 36 4 (11.2%) 16 (44.4%) 16 (44.4%) 0.2

Histology (adenocarcinoma) Undifferentiated 39 7 (17.9%) 23 (59.0%) 9 (23.1%) Differentiated 41 5 (12.2%) 19 (46.3%) 17 (41.5%) 0.15 Abbreviations: IR, immunoreactivity; TNM, tumor, node, metastasis. aPo0.05 was considered as statistically significant. specimens (32.5%). MGr1-Ag expression in the tumors was and MKN45/MGr-si cells in nude mice. Compared with scr-siRNA- significantly higher than that in normal mucosa (Figure 1a). To transfected cells, the injection of SGC7901/MGr1-si or MKN45/ further confirm this observation, four pairs of human GC and MGr-si cells led to a significantly decreased tumor size (Figures 3a corresponding noncancerous tissue specimens with known and b). Immunohistochemical analysis showed that MGr1-Ag expression of MGr1-Ag in immunohistochemistry assay were protein expression in the MGr1-Ag-siRNA-transfected cell group analyzed by western blot and RT-PCR. Consistently, MGr1-Ag was decreased compared with that in the control groups expression was higher in GC tissues than in noncancerous region (Figure 3c). Both in vitro and in vivo assays suggested that of GC tissues (Figures 1c and d). To evaluate the relationship knockdown of MGr1-Ag had the potential to inhibit the between MGr1-Ag expression and clinical outcome of GC patients, proliferation, growth and tumorigenicity of gastric cancer. we used a log-rank test with Kaplan–Meier estimates to determine whether MGr1-Ag is a significant prognostic factor for survival of MGr1-Ag siRNA inhibits cell cycle S-phase entry of SGC7901 and patients with surgically resected gastric carcinoma. The results MKN45 cells showed that the median of disease-specific survival was 36.1 months for cancer patients with low MGr1-Ag expression, and 25.6 To further investigate the mechanism by which MGr1-Ag inhibits months for moderate and 19.3 months for high MGr1-Ag GC cell growth, we studied the effects of MGr1-Ag expression on expression (Figure 1b). Western blotting and RT-PCR also showed the cell cycle by fluorescence-activated cell sorter analysis. The that MGr1-Ag protein and mRNA levels were higher in the GC cell results of the cell cycle showed that at 24 h after the release of lines SGC7901, MKN-45, AGS, and MKN-28 compared with synchronized cultures, 24.6% of SGC7901- MGr1-Ag siRNAs were in immortal gastric epithelial mucosa cell GES-1 (Figures 1e and f). S phase compared with 13.2% of scr-siRNA-transfected cells. The results were similar in MKN45-transfected cells (P o0.05, Figure 4a). Taken together, these data indicate that the siRNA targeting MGr1-Ag knockdown inhibits GC cell proliferation and growth in vitro and tumorigenicity in vivo MGr-Ag exhibited an inhibition of S-phase entry in GC cells. In order to explore the underlying molecular mechanism of To explore the effect of MGr1-Ag inhibition on proliferation of how MGr1-Ag affects the cell cycle distribution, we detected the GC cells, the MGr1-Ag-specific siRNA vectors were infected with expression of cell cycle-related molecules such as cyclin D1, p27, SGC7901 and MKN-45 cells using a lentivirus-based delivery cdk2 and p21in MGr1/si-transfected SGC7901 and MGr1/ system. First, fluorescence microscopy was used to observe si-transfected MKN45 cells by western blot analysis. The results infection efficiency (Figure 2a). Then, western blot assays showed that the expression of cyclin D1 expression was down- confirmed that MGr1 siRNA effectively downregulated MGr1-Ag regulated whereas that of p27 was upregulated (Figures 5a and b) expression in SGC7901 and MKN-45 cells (Figure 2b). in these cells. However, the cdk2 and p21 were not affected by We then tested the effect of MGr1-Ag siRNA on the cell viability inhibition of MGr1-Ag (data not show). Therefore, we may infer of SGC7901 and MKN45 cells in culture. As shown in Figures 2c that the induction of G1- to S-phase arrest of GC cells by knocking and d, MGr1-Ag siRNA significantly inhibited cell growth of down MGr1-Ag was partly mediated by downregulation of cyclin SGC7901 and MKN45 cells as compared with scr-siRNA-transfected D1 and upregulation of p27 expression. cells. To determine the effect of MGr1-Ag on the colony-forming ability of GC cells, we performed in vitro soft agar assay (Figures 2e and f). The results showed that downregulation of MGr1-Ag could Inhibition of MGr1-Ag by siRNA induces cell apoptosis in SGC7901 decrease cell growth in soft agar (Po0.05). and MKN45 cells Furthermore, in vivo subcutaneous tumor formative assay was The effect of MGr1-Ag siRNA on the apoptosis of SGC7901 and adopted to examine the proliferative ability of SGC7901/MGr1-si MKN45 cells was investigated by flow cytometry (Figures 4b

& 2014 Nature America, Inc. Cancer Gene Therapy (2014), 355 – 363 MGr1-Ag can promote gastric cancer proliferation L Liu et al 358

Figure 1. The MGr1-Ag expression in gastric cancers (GC). (a) Expression of MGr1-Ag as determined by immunohistochemical staining with MGr1-Ag antibody. (a) Noncancerous region of gastric cancer; (b, c) primary site of gastric cancer; (b) Kaplan–Meier plots of overall survival in patients with gastric cancer. (c, d) Whole-cell protein and RNA extracts prepared from four paired noncancerous regions (N) and gastric cancer tissues (T) by western blot and reverse transcription-PCR (RT-PCR). ##Po0.01 vs noncancerous region of gastric cancer. (e, f) Whole-cell protein and RNA extracts prepared from four gastric cancer cells and immortal gastric epithelial mucosa cells GES-1 by western blot and RT-PCR. **Po0.01 vs gastric epithelial mucosa cells GES-1.

and c). It was found that 13.2 or 11.5% of the SGC7901 or MKN45 cells compared with src-siRNA-transfected counterparts, suggest- cells transfected with src-siRNA were observed to be apoptotic, ing that the apoptotic effect of MGr1-Ag may be partly mediated whereas signiﬁcantly more apoptotic cells were observed in those by these Bcl-2 family proteins and caspase-3 (Figures 5a and b). transfected with MGr1-Ag siRNA in SGC7901 (26.1%) or MKN45 (25.2%). These data suggested that knockdown of MGr1-Ag by lenti-siRNA/MGr1-Ag speciﬁcally induced apoptosis of the LM-binding site of MGr1-Ag mediates the regulatory effect of MGr1-Ag-overexpressing GC cell lines SGC7901 and MKN45. MGr1-Ag on proliferation of gastric cancer In order to explore the underlying molecular mechanism of how A previous study reported between 161–179 and 205–229 MGr1-Ag affected cell apoptosis, we analyzed the Bcl-2 family sequence of MGr1-Ag as LM-binding site.20 To investigate mediators of apoptosis, such as Bcl-2 and Bax, as well as caspase-3 whether the region of MGr1-Ag might contribute to the via western blot analysis. We found decreased Bcl-2 and increased proliferation effect of MGr1-Ag in gastric cancer, two deletion Bax and caspase-3 expression in lenti-siRNA/ MGr1-Ag-transfected mutant vectors of MGr1-Ag were constructed, including

Cancer Gene Therapy (2014), 355 – 363 & 2014 Nature America, Inc. MGr1-Ag can promote gastric cancer proliferation L Liu et al 359

Figure 2. Lentivirus vectors for the MGr1-Ag small interfering RNA (siRNA) were constructed and shown to be specific and potent for silencing MGr1-Ag expression in the SGC7901 and MKN45 gastric cell lines. (a) To observe the infection efficiency of MGr1-Ag siRNA (MGr-si) and scramble siRNA (Src-si) by fluorescence microscope. (b) Western blot analysis of MGr1-Ag protein expression in SGC7901 and MKN45 cells infected with the lenti-siRNA/MGr1-Ag and src-siRNA. (c, d) Lenti-siRNA targeting MGr1-Ag depressed the growth curves of SGC7901 and MKN45 cells compared with the scr-siRNA and uninfected parental cells, as determined using a Cellomics Arrayscan. (e, f) Colony formation assays showed that the lenti-siRNA/MGr1-Ag inhibited the number of cells and clones when compared with the scr-siRNA and uninfected parental cells. **Po0.01 vs uninfected parental cells and transfection with scr-siRNA vector cells.

& 2014 Nature America, Inc. Cancer Gene Therapy (2014), 355 – 363 MGr1-Ag can promote gastric cancer proliferation L Liu et al 360

Figure 3. Local injection of lenti-siRNA/MGr1-Ag-infected SGC7901 and MKN45 cells into mice resulted in inhibition of tumor growth in vivo. (a) The tumor size of the lenti-siRNA/ MGr1-Ag-transfected cell group was signiﬁcantly decreased compared with that of the control cell group. (b) Tumor growth curves showed a signiﬁcant growth tendency in the scr-siRNA-transfected cell group, whereas tumor growth in the lenti-siRNA/ MGr1-Ag-transfected cell group was clearly inhibited. **Po0.01 vs transfection with scr-siRNA vector cells. (c) MGr1-Ag protein expression in the lenti-siRNA/MGr1-Ag-infected cell group decreased in vivo compared with that in the other groups, as determined by immunohistochemistry; IgG was used as an internal control. SiRNA, small interfering RNA.

MGr1-AgDN and MGr1-AgDB, with deletion of N-terminal domain than those of control cells. Similarly, the results showed that (13–161) and LN-binding domain (13–230) in MGr1-Ag/37LRP, deletion of LN-binding domain could significantly decrease cell respectively (Figure 5c). We further transfected SGC7901 cells with growth in soft agar (Figure 5g). these two deletion mutant vectors and the stable cell lines were obtained and designated as SGC7901/MGrDN, SGC7901/MGrDB and SGC7901/MGr (transfected with full length of MGr1-Ag), DISCUSSION respectively. We examined the expression of deletion mutants of Several studies reported by our group demonstrated that MGr1- MGr1-Ag in these stable cell lines. To exclude the interference of Ag/37LRP plays an important role in GC progression. However, the internal MGr1-Ag in SGC7901 when performing western blot, the exact mechanism is not clear and little clinical data that are directly expression of external MGr1-Ag deletion mutants was detected by associated with prognosis are available for this protein. In this anti-6His antibody. As shown in Figure 5d, all the 6His-tagged study, we presented the first evidence that MGr1-Ag/37LRP had mutants of MGr1-Ag/37LRP could be detected whereas no signal high expression in GC tissues and cells, and the high expression of was found in the control cell transfected with empty vector. MGr1-Ag/37LRP was consistent with the decreased median survival We also investigated the effect of LN-binding region on cyclin time of GC patients that suggested it might be used as an D1, p27, Bcl-2 and Bax expression. The results derived from independent prognostic factor in gastric cancer. western blot showed that cyclin D1 and Bcl-2 could be We also found that lentivirus-based delivery system inhibition of upregulated by MGr1-Ag or MGr1-AgDN transfection. They were MGr1-Ag expression significantly inhibited GC cell growth in vitro. downregulated by MGr1-Ag DB transfection. However, p27 and Moreover, inhibition of MGr1-Ag expression also significantly Bax expression was decreased in gastric cancer cells trasfection decreased GC cell tumorigenicity in nude mice transplant with MGr1-Ag or MGr1-AgDN transfection cultured on laminin tumor models. We observed a significantly decreased S-phase (1mg/cm À 2). And they were increased expression in MGr1-AgDB population in lenti-siRNA/ MGr1-Ag-transfected SGC7901 and transfection (Figure 5d). MKN45 cells, and this blockage to S-phase entry was connected To further explore the effect of LN-binding region of MGr1-Ag with the downregulation of cyclin D1 and upregulation of p27 on cell proliferation, we then tested the deletion mutants of LN- expression. This result indicated that knockdown of MGr1-Ag binding region on the cell viability of SGC7901 cells in culture. As expression inhibited GC cell proliferation regulated by cyclin D1 shown in Figures 5e and f, SGC7901-transfected MGr1-AgDB, with and p27 to activate expression of genes necessary for entering deletion of LN-binding domain, significantly inhibited cell growth into the S phase. as compared with control cells. When the growth curves of these Our previous studies revealed that the expression of Bcl-2 cell lines were compared in a medium containing 10% fetal calf family proteins was associated with hypoxia-inducible factor 1 serum, the curves for MGr1-AgDB cells were significantly lower (HIF-1)-dependent cell growth and apoptosis in SGC7901 cells.21

Cancer Gene Therapy (2014), 355 – 363 & 2014 Nature America, Inc. MGr1-Ag can promote gastric cancer proliferation L Liu et al 361

Figure 4. The effects of lenti-siRNA/MGr1-Ag on cell cycle and cell apoptosis as determined by ﬂow cytometric analysis. (a) Flow cytometry analysis showed that knockdown of MGr1-Ag expression via the lenti-siRNA/MGr1-Ag in SGC7901 and MKN45 cells induced G1-phase arrests and decreased the S-phase population of the cells, indicating disruption of cell cycle progression. (b, c) Flow cytometry analysis showed that knockdown of MGr1-Ag increased cell apoptosis in SGC7901 and MKN45 cells. **Po0.01 vs transfection with scr-siRNA vector cells. SiRNA, small interfering RNA.

& 2014 Nature America, Inc. Cancer Gene Therapy (2014), 355 – 363 MGr1-Ag can promote gastric cancer proliferation L Liu et al 362

Figure 5. The effects of different deletion mutants of MGr1-Ag on proliferation in gastric cancer. (a, b) Cell cycle-related molecules were assayed via western blot analysis. Knockdown of MGr1-Ag resulted in downregulation of cyclin D1 and upregulation of p27 expression in SGC7901 and MKN45 cells. Western blot analysis of Bcl-2 family mediators of apoptosis showed that knockdown of MGr1-Ag resulted in downregulation of Bcl-2 and upregulation of Bax in lenti-siRNA/MGr1-Ag SGC7901 and MNK45 cells. (c) Full length of MGr1-Ag/37LRP and deletion mutants MGr1-AgDN (13–161) and MGr1-AgDB (13–230) were constructed. (d) The expression of MGr1-Ag/37LRP in cells was evaluated by western blot with anti-6His antibody (Ab). The expressions of Bax, Bcl-2, cyclin D1 and p27 were also examined by western blot. b-Actin was used as an internal control. (e, f) Deletion mutants MGr1-AgDB (13–230) depressed the growth curves of SGC7901 cells as compared with the SGC7901/MGr and SGC7901/con cells as determined using Cellomics Arrayscan. (g) Colony formation assays showed that deletion mutants MGr1-AgDB (13–230) decrease the clones when compared with the SGC7901/MGr and SGC7901/con cells. **Po0.01 vs SGC7901/con cells and transfection with MGr1-Ag vector cells. SiRNA, small interfering RNA.

In addition, our further study confirmed that MGr1-Ag was a novel the structure of MGr1-Ag/37LRP in promotion of proliferation is HIF-1 target gene in gastric cancer.22 However, the mechanism of barely understood. The present study suggested that LM-binding the involvement of MGr1-Ag in proliferation and apoptosis is still domain of MGr1-Ag/37LRP was a necessary region for down- unknown. These studies found that depletion of MGr1-Ag could regulation of cyclin D1 and upregulation of p27 expression, as well decrease Bcl-2/Bax ration and upregulate caspase-3 expression, as to downregulate Bcl-2 and upregulate Bax. These data demonstrating that Bcl-2 family members and caspase-3 may suggested that this region is a putatively function domain of contribute to the apoptosis of GC cells following knockdown of MGr1-Ag in prompting proliferation in gastric cancer. MGr1-Ag. These results suggested that knockdown of MGr1-Ag Several lines of evidence suggest that cytoskeleton rearrange- expression may be essential for blocking cell proliferation and ment elevates FAK activity, subsequently activating the phospha- survival as well as promotion of cell apoptosis in GC cells. tidylinositide 3-kinase/AKT and mitogen-activated protein kinase Structure analysis of MGr1-Ag/37LRP revealed that it contained signaling pathways to perform the anti-apoptotic function. a transmembrane domain (amino acids 86–101), N-terminal Although a definitive signaling pathway remains elusive, it is intracellular domain and three 37LRP-interacting sequences: the notable that melanocyte differentiation antigens modulate the most C-terminal 53 amino acids containing four acidic TEDWS activities of a variety of kinases, such as protein kinases A and C, FAK repeats; a putative helical stretch between amino acids 205 and and phosphatidylinositide 3-kinase.26–28 We will explore the role of 229; a heparin-sulfate-dependent LM-binding region from 161 to the pathway in MGr1-Ag- and its LN-binding domain induced the 180.23 It is interesting to note that the same regions (161–180) of effect of anti-apoptosis and cell cycle arrest in the future study. MGr1-Ag/37LRP correspond to one of LM-binding sites.24 It was In summary, our results indicate that knockdown of MGr1-Ag reported that 37LRP also acts as a receptor for virus, and is expression can inhibit GC proliferation in vitro and in vivo.In involved with internalization of the prion protein.25 At present, addition, we confirmed that MGr1-Ag promoted GC proliferation

Cancer Gene Therapy (2014), 355 – 363 & 2014 Nature America, Inc. MGr1-Ag can promote gastric cancer proliferation L Liu et al 363 partly by upregulating cyclin D1 and Bcl-2 and inhibiting p27 and 12 Hengst L, Reed SI. Translational control of p27Kip1 accumulation during the cell Bax. Further study demonstrated that the effect of MGr1-Ag on cycle. Science 1996; 271: 1861–1864. proliferation in GC is dependent on the LM-binding region. In 13 Annabi B, Currie JC, Bouzeghrane M, Dulude H, Daigneault L, Garde S et al. conclusion, our present study confirmed that MGr1-Ag might be Contribution of the 37-kDa laminin receptor precursor in the anti-metastatic used as an independent prognostic factor and a potential PSP94-derived peptide PCK3145 cell surface binding. Biochem Biophys Res therapeutic target for gastric cancer. Commun 2006; 346: 358–366. 14 Jaseja M, Mergen L, Gillette K, Forbes K, Sehgal I, Copie V. Structure-function studies of the functional and binding epitope of the human 37 kDa laminin receptor precursor protein. J Pept Res 2005; 66: 9–18. CONFLICT OF INTEREST 15 Sun L, Shi Y, Guo C, Yao L, Lin T, Du J et al. Regulation of multidrug resistance by The authors declare no conflict of interest. MGr1-antigen in gastric cancer cells. Tumour Biol 2006; 27: 27–35. 16 Liu L, Zhang H, Sun L, Gao Y, Jin H, Liang S et al. ERK/MAPK activation involves hypoxia-induced MGr1-Ag/37LRP expression and contributes to apoptosis resis- ACKNOWLEDGEMENTS tance in gastric cancer. Int J Cancer 2010; 127: 820–829. 17 Liu N, Bi F, Pan Y, Sun L, Xue Y, Shi Y et al. Reversal of the malignant phenotype of This study was supported by Grants (no. 81101765 and no. 81272349) from the gastric cancer cells by inhibition of RhoA expression and activity. Clin Cancer Res National Nature Science Foundation of China. 2004; 10(18 Pt 1): 6239–6247. 18 Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D. 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& 2014 Nature America, Inc. Cancer Gene Therapy (2014), 355 – 363